[Effect of signal peptide optimized by site-directed mutagenesis on the function of chimeric antigen receptor T cells].

Signal peptides (SP) are involved in regulating the secretion level and transmembrane translocation of chimeric antigen receptors (CAR), which is crucial for CAR-T cells. This study aimed to optimize the SP sequence by site-directed mutagenesis and investigate its impact on the killing function of CD19-CAR-T. Firstly, CAR vectors targeting CD19 containing wild-type SP (SP-wtY) or two mutant SP (SP-muK or SP-muR) were constructed using gene synthesis and molecular cloning techniques. The successfully constructed vector was packaged with lentivirus, and T cells were infected. The transfection efficiency of T cells was detected by flow cytometry, while the killing effect on target cells was assessed using the calcein release method. The secretion levels of cytokines interferon-γ (IFN-γ) and interferon-α (TNF-α) were measured using enzyme linked immunosorbent assay (ELISA). The results showed that successful construction of recombinant lentivirus plasmids with wild type and signal peptide mutation. After the transferring the lentivirus into T cells, the transfection efficiency of CD19-CAR carrying three signal peptides (SP-wtY, SP-muK, or SP-muR) were 33.9%, 35.5%, and 36.8%, respectively. Further killing assay showed that the tumor-killing effect of SP-muR cells was significantly higher than that of SP-muK and SP-wtY cells. When the ratio of effector to target was 10:1, the secretion levels of cytokines IFN-γ and TNF-α of CAR-T cells of the SP-muR group were significantly higher than those in SP-muK and SP-wtY groups. In summary, this study revealed that increasing the N-terminal positive charge of the signal peptide can improve the expression efficiency of CAR and promote the killing of CD19+ target cells. These findings provide a scientific basis the optimization and clinical application of CAR structure.

Expression and purification of recombinant human serum albumin fusion protein with VEGF165b in Pichia pastoris.

VEGF165b is an endogenous transcriptional splice variant of VEGF and has been shown to have a therapeutic potency as an anti-cancer agent. In this report, a fusion gene consisting of a human VEGF165b and a human albumin (HSA) gene was constructed and then inserted into a pPIC9k vector. The recombinant fusion protein, rhHSA-VEGF165b, was over expressed in the methylotrophic yeast Pichia pastoris under the control of AOX1 promoter. After induction with methanol, the expression level of rhHSA-VEGF165b was 275 mg/L in broth. The fusion protein rhHSA-VEGF165b was purified to more than 95% purity by using Blue Sepharose Fast Flow and SP Sepharose Fast Flow. Biological activity of the prepared rhHSA-VEGF165b was characterized by transwell migration assay, retaining about 9% of that of unmodified rhVEGF165b on a molar basis. Data from mice show that the serum half-life time of rhHSA-VEGF165b was nearly 20 times longer than that of rhVEGF165b.